Our continuous efforts in animal genomics to identify genes and their functions have made it possible to generate transgenic animals. Transgenic animals have been created great commercial value in industries such as biomedicine and agriculture. Various techniques such as microinjection, viral transfection, sperm vector, application of embryonic stem (ES) cells and somatic cell nuclear transfer (SNCT) have also been developed to prepare transgenic animals or so called genetically-modified animals.
The microinjection method, which injects a DNA molecule into the male or female pronucleus of fertilized eggs (Harbers et al., Nature., 293(5833); 540-2, 1981; Brinster et al., Cell., 27; 223-231, 1981; Gordon et al., Proc Natl Acad Sci USA., 77(12); 7380-7384; Costantini et al., Nature., 294(5836); 92-94), has been widely employed to produce transgenic animals (Hammer et al., Nature., 315(6021); 680-683, 1985; van Berkel et al., Nat. Biotechnol., 20(5); 484-487, 2002; Damak et al., Biotechnology (NY), 14(2); 185-186, 1996). However, the efficiency of this technique in the production of transgenic animals is very low in that only 2-3% of the injected eggs give rise to transgenic offspring (Clark et al., Transgenic Res., 9; 263-275, 2000). The production of transgenic animals using the microinjection method is also a labor-intensive and costly procedure requiring large numbers of animals and facilities (Brink et al., Theriogenology, 53; 139-148, 2000). Another disadvantage of this technique is that it cannot control the integration sites and the copy numbers of the inserted genes. The resulting random integration sometimes leads to a low or nonspecific expression of the transgenes. It was further reported that the unregulated expression of certain transgenes sometimes cause lethality in the embryonic development (Wei et al., Annu Rev Pharmacol Toxicol., 37; 119-141, 1997).
The retrovirus-mediated method is also widely used in order to genetically manipulate animals (Soriano et al., Genes Dev., 1(4); 366-375, 1987; Hirata et al., Cloning Stem Cells., 6(1); 31-36, 2004). Under this technique, the desired gene sequences are introduced into the animal genomes by using virus-mediated vectors. Although viral transformation is more efficient than pronuclear injection, random insertion of the foreign genes and mosaicism are entailed due to multiple integrations (Piedrahita et al., Theriogenology, 53(1); 105-116, 2000). Additionally, the maximum size of the introduced genes is usually limited to approximately 7 kb and there is concerning of the potential interference caused by virally encoded proteins (Wei et al., Annu Rev Pharmacol Toxicol., 37; 119-141, 1997; Yanez et al., Gene Ther., 5(2); 149-159, 1998).
To circumvent the problems referred to above, a gene targeting technique that can insert or remove a DNA segment at a specific location was developed. The gene targeting technique was first applied to the mouse embryonic stem cells to study gene function. Mouse embryonic stem cells are now being used to introduce predetermined genetic modifications into embryos. A number of specific gene-targeted mice have been produced through the manipulation of mouse embryonic stem cells using the technique(Brandon et al., Curr Biol., 5(6); 625-634, 1995; Capecchi et al., Science, 244(4910); 1288-1292, 1989; Thompson et al., Cell, 56(2); 313-321, 1989; Hamanaka et al., Hum Mol. Genet., 9(3); 353-361, 2000; Thomas et al, Cell, 51(3); 503-512, 1987; te Riele et al., Proc. Natl. Acad Sci USA, 89(11); 5182-5132, 1992; Mansour et al., Nature, 336(6197), 348-352, 1988; Luo et al., Oncogene, 20(3); 320-328, 2001). The extension of this gene targeting method to other mammalian species, particularly livestock, could bring numerous biomedical benefits such as mass production of pharmaceutical proteins and animal disease models.
Until now, most recombinant therapeutic proteins have been produced by cell culture systems, which use cells such as yeast, bacteria or animal cells. However, it is difficult to produce proteins in large scale using cell culture systems because of the limited capacity and high cost. Furthermore, for some proteins, additional steps are required to introduce proper posttranslational modifications such as glycosylation, γ-carboxylation, hydroxylation and so on (Houdebine et al., Transgenic Res., 9(4-5); 305-320, 2000; Lubo et al., Transgenic Res., 9(4-5); 301-304, 2000).
Animal bioreactors that produce valuable or therapeutic proteins have been evaluated as efficient and cost-effective expression systems. In particular, the large-scale production of therapeutic recombinant proteins from transgenic animals is much more cost-effective compared to the cell culture system (van Berkel et al., Nat. Biotechnol., 20(5); 484-487, 2002). Recombinant proteins produced in animal milk were known to be post-translationally modified in a way very similar to the human counterpart proteins (Edmunds et al., Blood, 91(12); 4561-4571, 1998; Velander et al., Proc Natl Acad Sci USA., 89(24); 12003-12007, 1992; van Berkel et al, Nat. Biotechnol., 20(5); 484-487, 2002).
Cow's milk is composed of approximately 88% water, 3.3% protein and the remaining carbohydrates and fat. The caseins, comprising 80% milk protein, are divided into four groups, alpha S1, alpha S2, beta and kappa casein. Beta casein is the most abundant protein in milk and is expressed in a concentration of 10 mg/ml in bovine milk (Brophy et al., Nat. Biotechnology., 21(2); 157-162, 2003).
The somatic cell nuclear transfer (SCNT) technique is more efficient way to make transgenic animals compared to the microinjection method because almost all of the cloned animals derived from transformed somatic cells are transgenic. animals (Brink et al., Theriogenology, 53; 139-148, 2000). It is also possible to predetermine the sex of animals and create a genetically homogeneous herd in order to produce a uniform product (Lubo et al., Transgenic Res., 9(4-5); 301-304, 2000; van Berkel et al., Nat. Biotechnol., 20(5); 484-487, 2002).
Until now, ES cells and vector constructs for targeting a specific gene have been considered as prerequisite elements to generate gene-targeted animals. However, there is a limitation in the use of ES cells from large livestocks, although some studies have developed ES-like cells in pig and cattle (Doetschman et al., Dev Biol., 127(1); 224-227, 1988; Stice et al., Biol Reprod., 54(1); 100-110, 1996; Sukoyan et al., Mol Reprod Dev., 36(2); 148-158, 1993; Iannaccone et al., Dev Biol., 163(1); 288-292, 1994; Pain et al., Development, 122(8); 2339-2348, 1996; Thomson et al., Proc Natl Acad Sci USA., 92(17); 7844-7848, 1995; Wheeler et al., Reprod Fertil Dev., 6(5); 563-568, 1994).
Instead, the use of normal somatic cells as nuclear donor cells has been suggested as an efficient and practical method to produce transgenic cattle (Brophy et al., Nat. Biotechnol., 21(2); 157-162, 2003; Cibelli et al., Science, 280(5367); 1256-1258, 1998; Campbell et al., Nature, 380(6569); 64-66, 1996; Wilmut et al., Experientia, 47(9); 905-912, 1997; Denning et al., Cloning stem cells, 3(4); 221-231, 2001), suggesting the possibility that somatic cells instead of embryonic stem cells can be used for targeting specific genes.
With the application of SCNT technique, promoter regions of milk protein genes have been used to direct the expression of recombinant protein in the milk of transgenic large animals (Schnieke et al. Science, 278(5346); 2130-2133, 1997; Baguisi et al., Nat. Biotechnol., 17(5); 456-461, 1999; Brophy et al., Nat. Biotechnol., 21(2); 157-162, 2003). However, the use of this technique in farm animals is still not practical unless the problems of low expression and/or ectopic expression due to the random insertion of genes are solved. The ectopic expression of foreign protein causes early embryonic lethality and is particularly severe in nervous system, as most nervous system structures develop in late embryonic and early postnatal stages (Gao et al., Neurochem Res., 24(9); 1181-1188, 1999). To eliminate or reduce these side-effects, a new method that allows the foreign protein to be expressed only during the lactation period and strictly in the mammary gland have been invented (Houdebine et al., Transgenic Res., 9(4-5); 305-320, 2000). Gene-targeting, known as the introduction of site-specific modification into a genome by homologous recombination event, is a powerful tool for tissue-specific expression of recombinant proteins (Muller et al., Mech Dev., 82(1-2); 3-21, 1999; Clark et al., J Mammary Gland Biol Neoplasia., 3(3); 337-350, 1998).
The generation of a first knock-in ovine has opened the door to make it possible to produce therapeutic foreign protein-targeted large animals (McCreath et al., Nature, 405(6790); 1066-1069, 2000). The COLT-2 targeting vector which has homology regions for COL1A1 gene that is highly expressed in fibroblasts was developed, allowing the promoter-trap enrichment of gene-targeting events. AATC2 transgene comprised of human al-antitrypsin (AAT) complementary DNA within an ovine beta-lactoglobulin (BLG) expression vector was designed to direct expression in the mammary gland, having separate transcription unit. The amount of AAT secreted from targeted lambs was 37-fold more than that from the sheep with multiple and random integration of the genes (McCreath et al., Nature, 405(6790); 1066-1069, 2000). Thus, gene targeting is now regarded to be the most powerful method to produce large amount of therapeutic protein. However, the application of the promoter-trap targeting vector is limited to transcriptionally active genes in the somatic cells. In general, transcriptionally active genes are more amendable to gene targeting than silent genes because they have a higher frequency of homologous recombination (Kuroiwa et al., Nat. Genet., 36(7); 775-780, 2004).
Milk proteins are expressed in a tissue-specific manner in the mammary gland. An over-expression of foreign protein is possible without causing lethality in embryonic or post-natal development by manipulating a gene coding of the milk proteins. Among such proteins, beta-casein would be one of the best candidates since it is expressed abundantly. The bovine beta-casein exists as a single copy gene in total genome and is not known to be expressed in the somatic cells except for those in the mammary gland. The targeting of foreign genes into the beta-casein gene, which is not expressed in the normal somatic cells, cannot be carried out by vectors utilizing the promoter trap.
Based on this background, the present inventors have created targeting vector cassettes specific for the bovine beta-casein gene, vectors inserted with a foreign gene using the said cassettes, bovine somatic cell introduced with the said vector, and nuclear-transferred embryo with the said bovine cell. The present inventors found that the foreign gene was correctly targeted to the beta-casein gene of bovine genomic DNA and confirmed the targeting events with the said vector were highly efficient. Therefore, the present inventors have been completed transgenic cattle which could produce a large scale of desired therapeutic protein using the said bovine beta-casein gene targeting vector cassette.